Cisplatin accumulates in the inner ear cochlea via organic cation transporter 2 (OCT2) and causes ototoxicity. Lansoprazole, a proton pump inhibitor, ameliorated cisplatin-induced nephrotoxicity via inhibiting OCT2. In the present study, we investigated the protective effect of lansoprazole against cisplatin-induced ototoxicity. In the zebrafish study, we compared the effect of lansoprazole on cisplatin induced ototoxicity using in vivo fluorescence imaging of the hair cells stained with YO-PRO1. The fluorescence signals in hair cells in zebrafish treated with cisplatin dose-dependently decreased. Co-treatment with lansoprazole significantly suppressed the decrease of fluorescence signals in zebrafish treated with cisplatin. Knockout of a zebrafish homolog of OCT2 also ameliorated the reduction of fluorescence signals in hair cells in zebrafish treated with cisplatin. We then retrospectively analyzed the medical records of Mie University Hospital to examine the otoprotective effect of lansoprazole. The incidence rate of ototoxicity was significantly lower in patients co-treated with LPZ compared to those without lansoprazole. These results suggest that lansoprazole should suppress cisplatin-induced ototoxicity by inhibition of OCT2.